Validating the Hypoglycaemic and Hypotensive Roles of Salvia serotina (Chicken Weed) in Normal Healthy Sprague–Dawley Rats

Diabetes mellitus (DM) is an endocrine disease and is characterized by hyperglycaemia. Salvia serotina L. (chicken weed) has been used in traditional medicine to treat various ailments including DM. Aqueous, hexane, ethyl acetate, and methanol crude extracts of S. serotina L. were investigated for their anti-oxidant activities and hypoglycaemic and hypotensive effects in normal, healthy Sprague–Dawley rats using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, oral glucose tolerance test (OGTT), and the CODA noninvasive blood pressure system to determine systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), and heart rate (HR). The aqueous extract caused a free radical scavenging effect with an IC50 value of 10.2 ± 1.01 µg/mL versus vitamin C (9.42 ± 1.01 µg/mL). The extract lowered the blood glucose concentration at the 150 minute interval (5.00 ± 0.22 mM vs. 6.51 ± 0.33 mM; p = 0.004) and the 180 minute interval (4.77 ± 0.27 mM vs. 5.93 ± 0.0.30 mM; p = 0.015). The hexane extract gave significant hypoglycaemic activity at the 120 minute interval (4.54 ± 0.21 mmol/L vs. 5.50 ± 0.17 mmol/L; p = 0.005). The hexane extract also significantly lowered the SBP (132 ± 6 mm Hg; p = 0.014), DBP (106 ± 7 mm Hg; p = 0.034), and MAP (114 ± 7 mm Hg; p = 0.023) versus the controls SBP (156 ± 4 mm Hg), DBP (132 ± 8 mm Hg), and MAP (140 ± 6 mm Hg). Bioassay-directed purification of the hexane extract yielded 3,7,11-trimethyl-1,6,10-dodecatrien-3-ol (1), 3,7,11-trimethyl-2,6,10-dodecatrien-1-ol (2), and 5,22-stigmastadien-3β-ol (3) as active principles. Hence, S. serotina L. showed anti-oxidant, hypoglycaemic, and hypotensive effects in the rats and may have potential applications in the treatment of diabetes.


Introduction
e World Health Organization (WHO) global report on diabetes showed that the number of people living with diabetes mellitus has almost quadrupled to 422 million adults since 1980. e disease is characterized by hyperglycaemia and causes alterations in protein, lipid, and fat metabolisms. e three major types of diabetes are type 1, type 2, and gestational. Type 2 diabetes is lifestyle-related and accounts for about 90% of all cases [1]. Effective management of diabetes mellitus includes blood glucose monitoring, diet planning, medication regime, and control of blood pressure to reduce cardiovascular risks [2].
Reactive oxygen species (ROS) are formed by the incomplete reduction of oxygen during aerobic metabolism [3], and free radicals can arise from endogenous metabolic processes and inflammation [4]. In diabetes mellitus, the free radicals produced during inflammation can react with biomolecules such as nucleic acids, proteins, lipids, and carbohydrates causing damage and progression of the disease. e elevated blood glucose concentration causes more powerful oxidizing molecules to be formed when plasma proteins become glycated [5]. Anti-oxidants can protect the body from damage by reacting with harmful free radicals. Plant-based anti-oxidants are responsible for the ability of foods to combat chronic diseases such as coronary heart disease [6]. e main pharmacological agents used to treat type 2 diabetes are the sulfonylureas, such as glibenclamide, which promote insulin secretion, and biguanides, such as metformin, which discourage the output of glucose from the liver during fasting. Other agents such as the alpha-glucosidase inhibitors are useful to diabetics in the reduction of postprandial hyperglycaemia [7]. Due to the high cost of these drugs and their unpleasant side effects, there is always a need for new therapies. One option is the use of natural products from medicinal plants. One example is the triterpenoid stigmasterol that increases serum insulin levels in type 2 diabetic rats [8].
e hypoglycaemic activities of stigmast-4-en-3-one and the corresponding alcohol, stigmast-4-en-3-ol from the bark of Anacardium occidentale (Cashew) have been reported [9]. Plants from the Salvia genus have been investigated for their anti-cancer, anti-microbial, and hypotensive effects. Salvia serotina L. is commonly called chicken weed in Jamaican and is known for its blood purifying effects [10] and is also used in conjunction with glyburide for improved diabetes management [11].
In this study, we surmise that S. serotina L. may have potential application in the treatment of diabetes mellitus by imparting hypoglycaemic and hypotensive effects in rats. We sought to validate the use of this plant in folklore medicine as a beneficial agent in reducing glycaemia and question whether its hypoglycaemic effect is comparable to the known oral hypoglycaemic agents: metformin and glibenclamide. erefore, the main objectives were to investigate the antioxidant activity of S. serotina L., observe its hypoglycaemic and hypotensive effects in Sprague-Dawley rats, and, lastly, elucidate the active principles.

Plant Preparation and Extraction.
e aerial sections of S. serotina L. (chicken weed) were air-dried at room temperature. e plant was then milled to yield a finely ground material, which was weighed and packed into a glass column. e plant was then extracted sequentially using hexane, ethyl acetate, and methanol. e extracts were filtered, and the solvent was then evaporated in vacuo to yield hexane, ethyl acetate, and methanol crude extracts. e aqueous extract was prepared by heating the milled plant (40 g) in distilled water (400 mL) while gently stirring on a hot plate for 12 hours with intermittent heating at 45°C. e mixture was filtered through a sieve, and the filtrate was evaporated by gentle heating at 60°C to yield a dark brown viscous liquid. Further evaporation was allowed at room temperature until a brittle material resulted.
ese four extracts were kept refrigerated for biological assays, purification, and elucidation of active natural compounds.

Phytochemical Screening.
Phytochemical examinations were performed on the three crude extracts using standard methods described in the literature with minor modifications [12,13].

Anti-Oxidant Assay.
e method used was similar to that described by Doss and Pugalenthi in 2012 with minor modifications [14]. DPPH is a stable radical with oxidizing power that forms a purple solution with methanol. DPPH becomes reduced when it reacts with an anti-oxidant whereby it accepts a proton and the solution changes from purple to yellow [15]. e plant extracts or ascorbic acid were weighed (4 mg) and dissolved in methanol (4 mL) to make up a stock concentration of 1 mg/mL. e stock solutions were diluted serially to yield working concentrations of 200, 100, 50, 25, 10, and 1 µg/mL. 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) was weighed (9.86 mg) and added to methanol (250 mL) to make a standard solution at 0.1 mM.
Two milliliter of each stock solution at each concentration was pipetted into a clean glass tube, and 2 mL of the DPPH standard solution was added to the tube. Negative control was set up by combining methanol (2 mL) and DPPH solution (2 mL). e tubes were incubated in the dark at room temperature for 45 minutes. Absorbance was taken at 517 nm using a UV-Vis spectrophotometer.
A radical scavenging activity was expressed as a 1% scavenging activity and calculated using the following formula: e I DPPH % was plotted against the sample concentration, and a logarithmic regression curve was established in order to calculate the IC 50 value (inhibitory concentration), which represents the concentration of the sample (µg/ mL), which is needed to decrease the absorbance of DPPH by 50%. ey were maintained there under the supervision of the animal care staff and a veterinarian consultant. All animals were housed at room temperature under 12-hour cycles of dark and light and were fed tap water and Purina Laboratory Diet ad libitum. Each experimental group had 6 rats to allow for statistical analysis.

Oral Glucose Tolerance Test (OGTT).
e OGTT was performed using the methods described by Wilson and Islam in 2012 and Rao and Naidu in 2010 with minor modifications [16,17]. Normal, healthy S-D rats (250-400 g) fasted overnight for approximately 12 hours. A fasting blood glucose reading was taken by making an incision at the tip of the tail, and a drop of blood was placed onto the Accu-Chek Active glucose test strip that was inserted into the associated glucometer. e crude extracts (0.3 mL) were then administered orally at 200 mg/kg body weight (BW) or intravenously (IV) at 50 mg/kg BW. e five semipurified fractions TBHeFR1 to TBHeFR5 were administered intravenously at 30 mg/kg BW. Recrystallization of TBHeFR5 yielded TBHeFR5II, which was administered intravenously at 10 mg/kg·BW. Control rats were treated with 0.3 mL of dimethyl sulfoxide (DMSO) for IV administration or cooking oil for oral administration. Blood glucose readings were then taken at the 30 and 60 minute intervals. Immediately following, a glucose load (1.75 g/kg BW; 0.3 mL) was administered orally in warm distilled water. Blood glucose readings were taken at 30 minute intervals for 2½ hours.

Blood Pressure Test.
Haemodynamic parameters were determined using the CODA noninvasive blood pressure test and the CODA machine from Kent Scientific Corporation, USA [18]. Rats were trained by subjecting them to the experimental conditions prior to the actual blood pressure determination. Four parameters were observed: systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), and heart rate (HR). e occlusion pressure recording cuff (O-cuff) was placed around the tail of the animal. is was followed by the volume pressure recording cuff (V-cuff). Basal readings were taken for 5 minutes, and the cuffs were removed. e extract (0.3 mL) was then administered intravenously at 50 mg/kg BW. Control rats were treated with 0.3 mL of dimethyl sulfoxide (DMSO). Immediately following, the O-cuff and V-cuff were replaced, and SBP, DBP, MAP, and HR were recorded for 20 minutes.

Purification of the Hypoglycaemic Crude Extract.
e hexane extract of chicken weed indicated hypoglycaemic activity. Chromatography separations were carried out using standard techniques [19,20]. e hexane extract (5.13 g) was fractionated by column chromatography on silica gel (200-425 mesh, stationary phase height of 30 cm) usinggradient elution with hexane and dichloromethane as the mobile phase. Fractions were collected in 50 mL portions and analyzed on thin-layer chromatography (silica gel) using a solvent system of hexane:dichloromethane (2:3). A total of 43 fractions were obtained, and fractions with similar TLC (silica gel) profiles were grouped resulting in 5 main fractions (TBHeFR1 to TBHeFR5), and the solvent was removed under reduced pressure at 50°C using a rotary evaporator. e fraction TBHeFR1 (1.852 g) was an intense red-orange solid, TBHeFR2 (0.1568 g) an orange-coloured solid material, TBHeFR3 (1.057 g) a red-orange gel, TBHeFR4 (0.5272 g) a yellow-orange solid, and TBHeFR5 (1.794 g) a dark-green solid material with yields of 36%, 0.03%, 0.21%, 0.10%, and 35%, respectively. ese major fractions were bioassayed using the OGTT on normoglycaemic Sprague-Dawley rats in order to carry out a bioassay-guided isolation of the active principles. Both TBHeFR3 and TBHeFR5 revealed a lowering in blood glucose levels. TBHeFR5 formed colourless needles on standing and was recrystallized using cold hexane. A white crystalline substance (15.5 mg) was obtained and detected at 254 nm as a blue fluorescent band with an R f value of 0.1 when chromatographed with 7:1 hexane:ethyl acetate. TBHeFR3 gave a yellow-orange oil (TBHeFR3C, 0.4817 g) when chromatographed in an ethyl acetate:hexane (2:3) solvent system.

Structure Elucidation.
Structural elucidation was carried out using gas chromatography-mass spectrometry (GC-MS), 1 H-NMR, 13 C-NMR, FTIR spectroscopy, as well as melting point analysis. Chemical structures were created using ACD/Chem Sketch Software version 14.

Statistical Analysis.
Graphical data for inhibition assays were analyzed using GraphPad Prism version 7.03. Readings for the OGTT were obtained in duplicates, and means were calculated. All readings for the haemodynamic parameter tests were collected and averaged for each 5 minute interval. Values were reported as mean ± standard error of the mean. Data were analyzed using Statistics Package for the Social Sciences (SPSS) version 22 and Student's t-test with p < 0.05 considered to be significant.

Phytochemical Screening.
e active crude hexane extracts contained terpenoids, steroids, and tannins (Table 1). e more polar solvents extracted a wider range of phytochemicals.

Anti-Oxidant Assay.
e most potent radical quenching occurred when a concentration of 25 µg/mL of the extract or vitamin C was administered ( Figure 1). e anti-oxidant activity of the aqueous extract was most similar to vitamin C. e IC 50 value for the aqueous extract was 10.21 ± 1.01 µg/mL while vitamin C had the IC 50 value of 9.42 ± 1.01 µg/mL. Both the aqueous and methanol extracts were expected to display an anti-oxidant effect since they contain phytochemicals such as flavonoids, as reported in Table 1, which are popularly known for their anti-oxidative properties [21]. e methanol extract gave an IC 50 value of 24.22 ± 1.05 µg/mL when compared with vitamin C. e methanol and aqueous extracts contained the same set of phytochemicals (Table 1). However, the methanol extract required a higher concentration than the aqueous extract in order to impart a radical scavenging activity that was similar to vitamin C. e less polar ethyl acetate and hexane extracts showed the lowest radical scavenging potential with IC 50 values of 63.28 ± 1.11 µg/mL and 94.00 ± 1.02 µg/mL, respectively.

Oral Glucose Tolerance Test (OGTT).
e aqueous extract caused a significant decrease in the blood glucose concentration when compared to metformin and glibenclamide ( Figure 2) at the 150 and 180 minute intervals (5.01 ± 0.22 vs. 6.51 ± 0.33; p � 0.004, and 4.77 ± 0.27 vs. 5.93 ± 0.30; p � 0.015), respectively. e ethyl acetate and methanol crude extracts imparted an effect similar to the control, but the hexane extract indicated a drastic decrease in the blood glucose from the 90 min interval at 6.72 ± 0.16 mM to 5.01 ± 0.28 mM at the 180 min interval (Figure 3). Hence, an oral dose-dependency test was carried out using the hexane extract at concentrations of (100, 200, 300, and 400) mg/kg BW. e dose-dependency test showed that the hexane crude extract was most effective when administered at 200 mg/kg BW. An intravenous administration was also performed (Figure 4) where the crude hexane extract indicated a significant lowering of the blood glucose concentration at the 120 to 150 min time intervals.
Purification of the active hexane extract was performed using chromatography techniques. Based on the OGTT of the semipurified fractions, the most effective fraction was TBHeFR3 that showed hypoglycaemic activity throughout the postprandial region ( Figure 5). e most significant hypoglycaemic activity was observed at the 120 min interval when compared with the control (4.63 ± 0.26 mM vs. 5.50 ± 0.17 mM).
TBHeFR5 was recrystallized to give TBHeFR5II that was administered intravenously ( Figure 5). e results showed that as the sample was purified, the hypoglycaemic effect became more significant. ere was a decrease in the blood glucose concentration in both the fasting and postprandial regions, significantly at the 60 minute interval when compared with the DMSO control (3.78 ± 0.14 mM vs. 4.70 ± 0.25 mM; p � 0.01). us, indicating that TBHeFR5II (stigmasterol) was responsible for the hypoglycaemic effect observed.

Blood Pressure.
e hexane crude extract displayed the most effective hypotensive effect in the rats causing a lowering of the SBP (p � 0.01 and p � 0.02), DBP (p � 0.03       Table 2). However, there was no effect on heart rate ( Figure 6).
is fraction yielded a white crystalline solid after recrystallization, TBHeFR5II, which gave a purple colour with ammonium molybdate and sulfuric acid spray, indicating the presence of a sterol. Detailed 1 H-NMR, 13 C-NMR, and IR spectral analysis, with a melting point value of 134°C that coincides with the value  e Scientific World Journal from the literature of 135°C [22], indicated the presence of the steroid 5,22-stigmastadien-3β-ol (2), commonly called stigmasterol (Figure 7).

Phytochemical Analysis of Salvia serotina L. Extracts.
e main objectives of the study were to investigate the effects of S. serotina L. (chicken weed) on the blood glucose concentration and blood pressure in normal Sprague-Dawley rats, observe the anti-oxidant potency of the  e Scientific World Journal crude extracts, and identify the active hypoglycaemic principles. S. serotina contained glycosides, saponins, flavonoids, terpenoids, tannins, and steroids based on the phytochemical analysis (Table 1). It is the presence of such phytochemicals in herbs, fruits, and vegetables that is responsible for the health benefits provided by these plants [26,27], and so the active principles can serve as potential therapies in diabetes.

Anti-Oxidant Activities of Salvia serotina L. Extracts.
e hyperglycaemia that precedes frank diabetes mellitus causes oxidative stress [28] due to the formation of advanced glycated end products (AGEs).
ese AGEs lead to the formation of reactive oxygen species (ROS) that are free radicals [29]. It was necessary to study the anti-oxidant effect of the various crude extracts as the phytochemical screening of the crude extracts (Table 1) indicated the presence of phenolics such as flavonoids, which have known anti-oxidative effects.
e aqueous extract showed a free-radical scavenging effect that was similar to the known anti-oxidant, vitamin C (Figure 2). e most potent radical quenching effect occurred when a concentration of 25 µg/mL of vitamin C was administered. At this concentration, the IC 50 value for the aqueous extract was 10.21 ± 1.01 µg/mL, while vitamin C had the IC 50 value of 9.42 ± 1.01 µg/mL. Both the aqueous and methanol extracts contained phytochemicals such as flavonoids (reported in Table 1), which are popularly known for their anti-oxidative properties. e difference in antioxidative property may be due to a greater concentration of the radical scavenging components being extracted in the more polar solvent, water, when compared with methanol. Anti-oxidant activity is dependent on the amount of total phenolic compounds, and so it can be ascertained that the aqueous extract had a larger variety of polyphenols than the methanol extract, which had an IC 50 value of 24.22 ± 1.05 µg/ mL. e ethyl acetate and hexane extracts showed weaker anti-oxidant effects because of their nonpolar properties, and so less of the radical scavenging compounds were removed in these extracts. Compounds that antagonize the effects of reactive oxygen species are effective in ameliorating oxidative stress and in the lowering blood pressure [30], and so this plant may have applications in the treatment of diabetic complications.

Hypoglycaemic Effects of Salvia serotina L. Extracts.
e hexane, ethyl acetate, methanol, and aqueous crude extracts were bioassayed by oral administration in Sprague-Dawley (S-D) rats at 200 mg/kg body weight (BW). e hexane crude extract caused a significant decrease in the blood glucose concentration at the 120 minute interval, and the aqueous crude extract gave a similar effect as metformin at the 150 and 180 minute intervals (Figure 1). e 120 minute interval is crucial in oral glucose tolerance tests, and so the hexane crude extract was used to carry out an oral dose-dependency test at concentrations of 100, 200, 300, and 400 mg/kg BW. e dosage dependency study highlighted 200 mg/kg BW as the most effective dosage. At higher levels of extract, the assimilation of harmful phytochemicals may cause an impairment in the insulin response and cause an increase in the blood glucose concentration as seen in Figure 3 since the excess amounts of polyphenols from plant species can be harmful [31]. Based on the results from the oral administration of the plant extracts, an intravenous administration of the plant extract at 50 mg/kg BW was carried out using DMSO as the carrier (Figure 4). ere was a significant lowering of the blood glucose concentration in the postprandial region at the 120 minute (4.54 ± 0.21 mM; p � 0.005) time interval when compared with the control (5.50 ± 0.17 mM). e intravenous mode of administration of the extract enabled the direct assimilation of the phytochemical into the bloodstream and retarded the possible degradation by enzymes in the gastrointestinal tract. It was also observable that the blood glucose concentration increased again after the 120 minute decline ( Figure 4) and so is also possible that sufficient amounts of the naturally isolated hypoglycaemic components did not enter the blood to cause any long-term effect during the three hours of the OGTT. When the extracts are in their crude states, there is a diversity of compounds present including glycosides that could contribute to a rise in the glycaemic peak. Additionally, the phytochemical compounds present could be only moderately absorbed [32] or slow-acting and may be more effective if the rats were treated continuously over a period of time to cause an accumulation in the blood. e semipurification of the hexane extract revealed TBHeFR3 that caused a lowering of the glycaemic peak and significantly lowered the blood glucose concentration at the

Hypotensive Effects of Salvia serotina L. Extracts.
Since hypertension is a major complication of diabetes mellitus, it was plausible to investigate the effect that a hypoglycaemic agent imposes on blood pressure. Salvia species have been used in ethnomedicine to reduce blood pressure by the inhibition of angiotensin-converting enzyme (ACE) [32]. Hypertension poses a great risk for cerebrovascular disease and coronary heart disease [33]. e CODA noninvasive blood pressure machine (Kent Scientific) was used to bioassay the hexane, ethyl acetate, and methanol crude extracts in normotensive S-D rats. e presence of saponins and flavonoids, which are marked vasodilators, was observed in the methanol and ethyl acetate extracts (Table 2), and so it was expected that these extracts would have conveyed a hypotensive effect, but this was not observed. e hexane crude extracts significantly lowered systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) from 10 minutes postextract administration, while there was no significant effect on heart rate ( Figure 6). Salvia species have been popular for various bioactivities such as anti-inflammatory and hypotensive effects in the treatment of nervous conditions and circulation disorders [34]. erefore, it is necessary to pursue further studies into the mechanism of action of S. serotina L. (chicken weed) in the lowering of blood pressure. In light of these observations, it was necessary to purify the hexane extract in order to identify the active compounds.

Bioassay Guided Purification of the Hexane Crude Extract.
e purification of the hexane extract afforded the plant sterol 5,22-stigmastadien-3β-ol, commonly called stigmasterol (Figure 7), as one of the active hypoglycaemic principles from the plant following chromatographic and spectroscopic analyses. e intravenous administration of stigmasterol (TBHeFR5II) crystals at 10 mg/kg BW (Figure 5) caused a reduction in the blood glucose concentration during the fasting and postprandial regions when compared with the DMSO control. Stigmasterol has beneficial bioactivities such as anti-oxidant, anti-hypercholesterolemic, and hypoglycaemic effects in diabetic rats [35]. e other two compounds of interest, Farnesol and Nerolidol, were present in fraction TBHeFR3. Oral administration of Farnesol caused a lowering of the glycaemic peak and reduced glycaemia in streptozotocin-induced type 2 diabetic mice by inhibition of alpha-glucosidase enzyme [36], while Nerolidol displayed various bioactivities both in vitro and in vivo by showing its anti-ulcer, anti-oxidant, and anti-inflammatory properties [37], thereby indicating the anti-diabetic properties of these active compounds. Overall, the results from Figures 3-5 show that the naturally isolated compounds seemed to be slow-acting, while the crude hexane extract was administered possibly due to the other components that were present. Some of these compounds could be hyperglycaemic in nature, and hence, they imparted an antagonistic effect on the active hypoglycaemic principle(s).
Additionally, the active compounds present may have taken a long time to be metabolized due to physicochemical properties such as lipophilicity. Overall, the results of this study validate the use of S. serotina L. as a blood purifier in folklore medicine due to the anti-oxidant, hypopglycaemic, and hypotensive effects that were observed.